Base-modified derivatives of 2′,5′-oligoadenylate and antiviral uses thereof

ABSTRACT

Antiviral compounds have the formula                    
     wherein 
     m is zero, 1, 2 or 3; n is from 1 to 8, preferably 1, 2 or 3; most preferably 1 or 2; 
     R is independently selected from the group consisting of                    
      provided that all R may not be                    
     R 1  is independently selected from the group consisting of hydroxyl and hydrogen; 
     R 2  is independently selected from the group consisting of oxygen and sulfur; 
     or water soluble salts thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase of PCT/US98/11095 pursuantto 35 U.S.C. 371, which claims the benefit of U.S. provisionalapplication Ser. No. 60/049.404, filed Jun. 12. 1997 and U.S.provisional application Ser. No. 60/052.027, filed Jul. 9. 1997.

FIELD OF THE INVENTION

This invention relates to synthetic analogues of naturally occurringantiviral 2′,5′-oligoadenylates wherein at least one of the nucleosideresidues is replaced with a modified nucleoside.

BACKGROUND OF THE INVENTION

The discovery of the 2′-5′ oligoadenylates is connected with the studyof the mechanisms of interferon action as the cellular response to virusinfection [2]. The 5′-triphosphate of the (2′-5′) oligoadenylate trimerplays a most important role in the antiviral mechanism induced byinterferon [3]. It is generally regarded that activation of RNase L by2-5A is key to the antiviral defense mechanisms. Interferon inducestranscription of the enzyme 2-5A synthetase which produces 2′,5′-linkedoligoadenylates upon activation of double-stranded RNA.

Previously, the only known biochemical effect of 2-5A is activation ofRNase L. This enzyme hydrolyzes mRNA and rRNA, thereby resulting ininhibition of protein synthesis. The activation of RNase L is transientunless 2-5A is continuously synthesized, since 2-5A is rapidly degraded.RNase L activation thus plays a critical role in inhibiting replication,and therefore in defending against infection by viruses.

Naturally occurring (2′-5′)oligoadenylates (both 5′-phosphorylated andunphosphorylated) have shown different kinds of biological activity[4][5]. Analogues of the natural (2′-5′)oligoadenylates have beensynthesized to achieve new approaches to antiviral and antitumoraltherapy [6-13]. Biological activities of 5′-phosphorylated(2′-5′)oligoadenylates are connected with the functioning of the(2′-5′)A system which leads to the inhibition of protein synthesis [3].The mechanism of action of unphosphorylated (2′-5′)oligoadenylates inmany cases is still unknown. Recently, certain sugar-modified trimers of(2′-5′)oligoadenylates were found to be inhibitors of HIV-1 reversetranscriptase (RT) [14-18].

SUMMARY OF THE INVENTION

The compounds of the present invention are useful in inhibiting viralinfections in plants and mammals.

The compounds and the water-soluble salts thereof are of the formulawherein

m is zero, 1, 2 or 3; n is from 1 to 8, preferably 1, 2 or 3; mostpreferably 1 or 2;

R is independently selected from the group consisting of

 provided that all R may not be

R₁ is independently selected from the group consisting of hydroxyl andhydrogen;

R₂ is independently selected from the group consisting of oxygen andsulfur.

Preferably, all R₁ are hydroxyl and all R₂ are oxygen.

According to one preferred embodiment of the invention, R is selectedfrom the group consisting of

According to another preferred embodiment of the invention, R isselected from the group consisting of

According to yet another preferred embodiment of the invention, the R ofthe 2′,3′-terminal nucleoside moiety is

Compounds of the invention include, for example, the following corecompounds, the 5′-mono, di-, and triphosphates thereof, andwater-soluble salts of any of them:

adenylyl-(2′-5′)-adenylyl-(2′-5′)-1-(β-D-ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide;

adenylyl-(2′-5′)-[1-(β-D-ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide]yl-(2′-5′)-adenosine;

1-(β-D-ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide]yl-(2′-5′)-adenylyl-(2′-5′)-adenosine;

adenylyl-(2′-5′)-adenylyl-(2′-5′)-N⁶-benzyladenosine;

adenylyl-(2′-5′)-N⁶-benzyladenylyl-(2′-5′)-adenosine;

N⁶-benzyladenylyl-(2′-5′)-adenylyl-(2′-5′)-adenosine; and

N⁶-benzyladenylyl-(2′-5′)-N⁶benzyladenylyl-(2′-5′)-N⁶-benzyladenosine.

The invention also comprises a method of treating viral infection inmammals or plants by administering an antivirally effective amount of acompound according to the above formula, or a water-soluble saltthereof. The invention further comprises an antiviral compositioncomprising such a compound or water soluble salt in combination with anagricultural carrier or pharmaceutical carrier.

DETAILED DESCRIPTION OF THE INVENTION

Replacement of the adenine moiety in the (2′-5′)oligoadenylate trimercore with 1H-1,2,4-triazole-3-carboxamide (TCA) or withN⁶-(benzylamino)purine (Ade^(Bn))-moieties has resulted in a new groupof inhibitors of HIV-1 replication. Trimer compounds of the presentinvention and their synthesis intermediates are numbered 1 through 28 asfollows for purposes of identification. Compound 29 is the authentic(2′-5′)oligoadenylate trimer core.

Base R R¹ R² 1 Ade^(Bn) H H H 2 Ade^(Bn) MeOTr H H 3 Ade^(Bn) MeOTr BzBz 4 Ade^(Bn) MeOTr H Bz 5 Ade^(Bn) MeOTr Bz H 6 Ade^(Bn) H Bz Bz 7 TCAH H H 8 TCA MeOTr H H 9 TCA MeOTr Bz Bz 10 TCA MeOTr Bz H 11 TCA H Bz Bz12 Ade^(Bz) H Bz Bz

Base¹ 13 Ade^(Bn) 14 TCA 15 Ade^(Bz)

Base Base¹ 16 Ade^(Bn) Ade^(Bz) 17 Ade^(Bn) Ade^(Bn) 18 Ade^(Bz)Ade^(Bn) 19 TCA Ade^(Bz) 20 Ade^(Bz) TCA 21 Ade^(Bz) Ade^(Bn)

Base Base¹ Base² 22 Ade^(Bn) Ade Ade 23 Ade Ade^(Bn) Ade 24 Ade AdeAde^(Bn) 25 Ade^(Bn) Ade^(Bn) Ade^(Bn) 26 TCA Ade Ade 27 Ade TCA Ade 28Ade Ade TCA 29 Ade Ade Ade

In the compounds of the present invention, each R₂ may be oxygen, eachR₂ may be sulfur, or R₂ may comprise a mixture of oxygen and sulfur toprovide a backbone of a 2′,5′-phosphodiester, a 2′,5′-phosphorothioateor a 2′,5′-mixed phosphorothioate/phosphodiester oligonucleotide,respectively.

The substitution of sulfur for oxygen in the 2′,5′-phosphodiesterbackbone referenced above, introduces chirality into the molecules andintroduces a new chemistry of the backbone. The core2′,5′-phosphorothioates exhibit increased resistance tophosphodiesterase and phosphatases and new biological activitiescompared to authentic 2-5A cores. The preparation of the2′,5′-phosphorothioates, including fully resolved enantiomers thereof,is disclosed in U.S. Pat. No. 4,924,624 and is incorporated herein byreference. A mixture of phosphorothioate and phosphodiester linkages ispossible in the same oligomer, providing molecules with a mixedphosphodiester/phosphorothioate backbone, as described inPCT/US95/10683, the entire disclosure of which is incorporated byreference.

While the preparation and examples that follow are directed to oligomersof a base-modified adenosine, the procedure described is equallyapplicable to the manufacture of oligomers comprising a base-modifiedcordycepin, or mixed chain of cordycepin and adenosine residues asdescribed in U.S. Pat. No. 4,859,768, the entire disclosure of which isincorporated by reference.

Chemical Synthesis

Syntheses of the compounds of the present invention may be achieved bythe phosphotriester method using a published approach [19]. SyntheticN⁶-benzyladenosine 1 and1-(β-D-ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide 7, obtained by thereaction of microbiological transglycosylation as described before [20],are converted into the corresponding selectively blocked nucleosides 2-6and 8-11 and corresponding nucleotides 13 and 14, respectively. Thus,treatment of 1 with monomethoxytrityl chloride (MeOTrCl) in pyridinegives the 5′-O-monomethoxytrityl derivative 2 (85%). Benzoylation of 2with benzoyl cyanide (BzCN) in MeCN [19] leads to a mixture of the2′,3′-di-O-benzyl 3, 2′-O-benzoyl 4, and 3′-O-benzoyl 5 derivativeswhich are isolated by column chromatography (CC) in 38, 13, and 46%yield, respectively. Treatment of 3 with a 2% solution of TsOH inCH₂CL₂/MeOh 7:3 affords 2′,3′-di-O-benzoyl-N⁶-benzyladenosine 6 in 96%yield. Similarly, 7 is converted into the 5′-O-monomethoxytritylderivatives 8-10 in 92, 16, and 57% yield, respectively. Detritylationof 9 leads to the 5′-OH derivative 11 in 78% yield. Furthermore, thereaction of the 3′-O-benzoylated compounds 5 and 10 with 2-chlorophenylbis (1H-1,2,4-triazol-1-yl)-phosphinate followed by subsequenttreatments with 2-(4-nitrophenyl)-ethanol (NpeOH) and then a solution of4-nitrobenzaldoxin in dioxane/H₂O/Et₃N 1:1:1, gives the correspondingnucleoside 2′-phosphodiesters 13 and 14 which are isolated by CC (silicagel) in 82 and 48% yield, respectively.

The compounds 6, 11, 13, and 14 and the corresponding adenosinederivatives 12 [21] and 15 [22] are then used in the syntheses of thenew. (2′-5′)oligonucleotide trimers 22-29. Condensation of2′,3′-di-O-benzoyl-N⁶-benzyladenosine 6 with the 2′-phosphodiester 15 inpyridine in the presence of a mixture of1H-triazole/2,4,6-triisopropylbenzenesulfonyl chloride (TpsCl) 3:1,followed by detritylation, leads to the 5′-OH dimer 16 which is isolatedby CC in 85% yield. Similar reaction sequences convert compounds 6 and13, 12 and 13, 11 and 15, and 12 and 14, into the corresponding 5′-OHdimers 17-20, isolated in 81, 87, 74, and 76% yield, respectively. Thesynthesis of 5′-OH dimer 21 has been described earlier [22].

The transformations of the dimers 16-20 to the trimer level is affordedthe same techniques consisting of a condensation step and followed bysuccessive treatment with 2% solution of TsOH, 0.5M DBU(1,8diazabicyclo[5.4.0]undec-7-ene)/(pyridine, and NH₃/MeOH,respectively, to remove the three different protecting groups. Finalpurification by CC(DEAE-cellulose)ion exchange gives the trimers 22-28in good to moderate yields.

Biological Utility

The compounds of the present invention may be combined with appropriatepharmaceutical or agricultural carriers to form an antiviralcomposition.

For pharmaceutical use, the compounds of the invention may be taken upin pharmaceutically acceptable carriers, such as, solutions,suspensions, tablets, capsules, ointments, elixirs and injectablecomposition and the like. They are administered to subjects sufferingfrom viral infection. The dosage administered depends upon the natureand severity of the infection, the disease stage, and, when administeredsystematically, the size and weight of the infected subject.

The compounds are generally administered in the form of water-solublesalts. Pharmaceutically acceptable water soluble salts include, forexample, the sodium, potassium or ammonium salts of the activecompounds. They are readily dissolved in water or saline solution. Thus,the preferred formulation for pharmacological use comprises a salinesolution of the desired compound in salt form. The formulation mayfurther contain an agent, such as a sugar or protein, to maintainosmotic balance. The salt form of the compound is preferred owing to therelatively high acidity (about pH 3) of the acid form of the compounds.

The compounds of the invention may be used as a treatment for humans andanimals from viral infectives such as Herpes simplex, rhinovirus,hepatitis and other infections of the hepatitis virus family, EpsteinBarr virus, measles virus, multiple sclerosis (which may be caused by aviral agent) and the various Human Immunodeficiency Viruses (“HIV”),such as HIV-1, which causes cutaneous T cell lymphoma, HIV-2, whichcauses Sezary lymphoma, and HIV-3, which is responsible for AcquiredImmune Deficiency Syndrome (“AIDS”). The compounds of the inventioninhibit HIV-1 induced syncytia formation.

The compounds may be applied topically to treat skin cancers caused byradiation, carcinogens or viral agents. Such skin cancers includecutaneous T-cell lymphoma, Sezary lymphoma, Xeroderma pigmentosium,ataxia telangiectasia and Bloom's syndrome. A sufficient amount of apreparation containing a compound of the invention is applied to coverthe lesion or affected area. An effective concentration of active agentis between about 10⁻³ M and 10⁻⁵ M, with 10⁻⁴ M being preferred.

The compounds of the present invention may also be used to treatplant-infecting viruses, particularly tobacco mosaic virus, and otherviruses which cause necrosis in turnips, cucumbers, orchids and in otherplants. Such viruses include, but are not limited to, tobacco veinmottling virus, vesicular stomatitis virus, vaccinia virus, turnipnecrosis virus, and cymbidium orchid virus.

The compounds may be administered effectively to plants by topicalapplication by abrasion of the leaf surface, aerosol spray, treatment ofthe soil, spraying, or dusting.

An effective antiviral composition may be formed by combining one ormore of the compounds of the invention with a carrier material suitablefor agricultural use. The active compound may also be administered byspraying insect vectors such as aphids, thrips and whiteflies whichcarry virus to plants. The dosage administered depends upon the severityof the infection.

The compounds of the invention may be applied to plant seeds prior togermination to control viruses contained in the germ plasm. The seedsmay be soaked in a solution of polyethylene glycol (“PEG”) containingone or more of the compounds. PEG brings the seeds to physiologicalactivity and arrest. The relative concentration of active compound toPEG depends upon the type of seed under treatment.

Plants may be effectively treated with an aqueous formulation containingfrom about 10⁻¹ to about 10⁻² M concentration of active ingredient. Thecompounds of the invention may be applied at very low concentrations. Aneffective amount of active ingredient on the plant surface is from about10⁻⁸ to about 10⁻¹² mole per cm² of plant surface area, with about 10⁻¹⁰mole to about 10⁻¹² mole per cm² being preferred. For the typicaltobacco plant, 10⁻⁵ M of compound is effective. At this rate, one poundof active ingredient is sufficient to treat 2×10⁸ tobacco plants.

For agricultural application, the compounds are advantageouslyadministered in the form of water-soluble salts, e.g. ammonium orpotassium salts. Sodium salts are generally avoided in treating edibleplants.

The compounds of the invention are readily dissolved in water,particularly at such low concentrations. Aqueous formulations foragricultural use may optionally contain a sticker and/or aUV-stabilizer. Such agents are well-known to those skilled in the art.Fatty acids (1%) are useful as spreader sticker agents. EffectiveUV-stabilizers include, for example, p-aminobenzoic acid.

For antiviral use in mammals, the compounds of the invention areadministered parenterally, such as intravenously, intraarterially,intramuscularly, subcutaneously or when administered as an anti-canceragent, intratumorally. The preferred route of administration forantiviral therapy is intravenous injection. The compounds of theinvention may be administered to mammals at very low concentrations. Theactual dosage administered may take into account the size and weight ofthe patient, whether the nature of the treatment is prophylactic ortherapeutic in nature, the age, health and sex of the patient, the routeof administration, the nature and stage of the affliction, and otherfactors. An effective daily dosage of active ingredient, based upon invivo studies involving other 2-5A analogues, is from about 0.25 g per 70kg of body weight (approximately 152 lbs) to about 2.5 g per 70 kg ofbody weight. The preferred daily dosage is about 0.5 g per 70 kg of bodyweight. Those skilled in the art should readily be able to deriveappropriate dosages and schedules of administration to suit the specificcircumstance and needs of the patient.

It is expected that an effective treatment regimen includesadministration of the daily dosage for two days. Treatment is continuedat least until the disease condition is substantially abated.

Preferably, the therapeutic end point is determined by testing for thecontinued presence of viral DNA. Such testing can be done by polymerasechain reaction (PCR) in which the presence of viral DNA is assayedaccording to conventional PCR. PCR primers of appropriate nucleotidesequences for amplification of viral DNA can be prepared from knownviral nucleotide sequences. To obtain DNA for testing, patientperipheral blood mononuclear cells are lysed with an appropriate lysingagent, such as NP-40.

Alternatively, testing for the continued presence of the virus can beperformed by an antigen-antibody assay using any of the known monoclonalor polyclonal antisera against a protein antigen of the target virusprotein coat. For example, an antigen-antibody assay may be employed todetect any of the protein antigen in the virus HIV protein coat, forexample, the gp120, p17 or p24. Moreover, the target antigen is notlimited merely to coat protein antigens. Antisera can be targetedagainst a suitable non-coat protein antigen, such as the HIV reversetranscriptase (RT) molecule. Monoclonal antibodies to HIV RT are known.Sobol et al., Biochemistry 1991, 30, 10623.

Additionally, testing for the presence of the infecting virus during orpost-treatment could be accomplished by an assay which assesses theviral load in the patient's blood stream. This can be done bydetermining syncytia formation. See procedure outlined in Henderson etal., Virology, 1991, 182, 186.

In addition to administration with conventional carriers, the compoundsof the present invention may be administered by a variety of specializedoligonucleotide or nucleic acid delivery techniques. 2-5A and itsanalogues have been successfully encapsulated in various encapsulatingmaterials, such as in unilamellar liposomes and delivered with the aidof monoclonal antibodies to cells, Bayard et al., Eur. J. Biochem.,1985, 151, 319. Reconstituted Sendai virus envelopes have beensuccessfully used to deliver RNA and DNA to cells, Arad et al., Biochem.Biophys. Acta. 1986, 859, 88. Moreover, the virus envelope is notlimited to Sendai virus, but could include encapsulation in anyretroviral amphotrophic particle. For example, an HIV envelope could beformed from any part or all of the outer protein coat of anon-infectious HIV particle. Such particles as gp 120 can be cloned byknown recombinant techniques. These techniques may be utilized forintroduction of the present 2-5A oligoadenylate derivatives into cells.It is further contemplated that the compounds of the invention may beadministered in the form of prodrugs in which lipophilic groups areattached to, for example, the 5′-terminal hydroxyl group of the corecompound.

Biological Studies

Three studies were performed to determine the antiviral activity of the(2′-5′)oligonucleotide derivatives of the present invention: (i)inhibition of HIV-1-replication, (ii) inhibition of HIV-1reverse-transcriptase (RT) activity, and (iii) activation of recombinanthuman GST-RNase L. All compounds were tested at a concentration of 300μM.

Inhibition of HIV-1 Replication

The infected centers assay as described by Henderson et al., Virology1991, 182, 186, was used to measure the ability of the trimer corecompounds of the invention to inhibit HIV-1 induced syncytia formation,an indicator of HIV-1 replication in T cells. Freshly isolatedperipheral blood lymphocytes (PBL) were treated with 2-5A trimer orderivatives for 2 hours and infected with HIV-1 strain IIIB at amultiplicity of infection of approximately 0.1. The infected PBL weremaintained in RPMI-1640 medium supplemented with 10% (v/v)heat-inactivated fetal calf serum at 37° C. in a humidified 5% CO₂ inair atmosphere. After 48 hours, the cells were washed twice in Hank'sbalanced salt solution, serially diluted and seeded into multiple wellsof a 96-well microtiter plate. Immediately, 2×10⁵ exponentially growingSup T1 cells were added to each well; Sup T1 cells readily form asyncytium with a cell which is productively infected with HIV-1. Thewells were examined daily for the presence of syncytia, using a tissueculture microscope. The first signs of syncytia formation can be seen in12 hours, with some complete syncytia developing by 24 hours. Finalresults were read at 72 hours. Each syncytium was counted as a singleinfected cell. The number of syncytia per seeded cell is determined andexpressed as an infected center per infected cell. The number ofsyncytia per 10⁴ cells was 121±16 for the control Sup T1 cells. The datais shown in Table 1. The mean of triplicate determinations is shown;variance did not exceed 5-10%.

Inhibition of HIV-1 Reverse-Transcriptase Activity

Sup T1 cells were treated with trimer core compound at 300 μM for 6hours and then infected with HIV-1 at a multiplicity of infection ofapproximately 0.1. At 96 hours post-infection, culture supernatant wasremoved and HIV-1 RT activity was assayed in triplicate as described byHenderson et al., Virology 1991, 182, 186. Briefly in this method, 25 μlof culture supernatant was added to a 50 μl cocktail containing 50 mMTris (pH 8.0), 20 mM dithiothreitol, 10 mM MgCl₂, 60 mM NaCl, 0.05%Nonidet p-40, 5 μg/ml oligodeoxythymidylic acid, 10 μg/mlpolyriboadenylic acid, 10 μM deoxythymidine triphosphate and 1mCi[α³²P]thymidine 5′-triphosphate. The mixture was incubated at 37° C. for2 hours. Fifty microliters of the cocktail were then spotted ontodiethylaminoethyl (DEAE) paper, dried, washed with 2×SSC solution (threetimes for 10 minutes each time) and 95% ethanol (two times for 5 minuteseach time), dried and exposed to radiographic film for 18 to 24 hours at−80° C. The filters were cut and final quantitation was determined byscintillation spectrometry.

The data for the HIV-1 RT activity is shown in Table 1 as a percent ofRT activity. Control values for RT activity ranged from 15,000 to 16,000dpm [α³²P] incorporated. The mean of duplicate determinations is shownin Table 1. Variance did not exceed 5-10%.

Activation of Recombinant Human GST-RNase L

Human recombinant RNase L was expressed in E. coli(DH5α) as a fusionprotein of glutathione-S-transferase (GST). Activation of humanrecombinant GST-RNase L was measured as the percent of poly(U)[³²P]pCphydrolyzed in the presence of authentic 2′,5′-oligoadenylate trimer coreor trimer core analog of the invention as described by Sobol et al., J.Biol. Chem. 1995, 270, 5963. The data is shown in Table 1 as the mean ofduplicate determinations. Variance did not exceed 5-10%.

Results of Biological Studies

TABLE 1 Inhibition of HIV-1-Replication and Biological Activities of(2′-5′) Oligonucleotide Trimers 22-29^(a)

Base Base¹ Base² Syn^(b) RT^(c) RNase L^(d) 22 Ade^(Bn) Ade Ade >1500 3337.4 23 Ade Ade^(Bn) Ade >1500 7.6 34.8 24 Ade Ade Ade^(Bn) >1500 16.7 025 Ade^(Bn) Ade^(Bn) Ade^(Bn) >1500 10.6 13.6 26 TCA Ade Ade 1.6 99.787.7 27 Ade TCA Ade 7.0 99.7 9.4 28 Ade Ade TCA 1.2 99.5 0 29 Ade AdeAde 3.0 50 ^(a)Compounds were tested at 300 μm. ^(b)Inhibition of HIV-1replication was determined by HIV-1-induced syncytia formation (foldreduction) for each compound. The number of syncytia/10⁴ cells was 121 ±16 for the control Sup T1 cells. The mean of triplicate determinationsis shown; variance did not exceed 5-10%. ^(c)Percent inhibition ofreverse-transcriptase (HIV-1 RT) activity. Control values for HIV-1 RTactivity ranged from 15000 to 16000 dpm incorporated. The mean ofduplicate determinations is shown; variance did not exceed 5-10%.^(d)The activation of recombinant human RNase L was measured as thepercent hydrolysis of poly(U)-3′-[³²P]pCp in the presence of the trimers22-29. The mean of duplicate determinations is shown; variance did notexceed 5-10%.

The TCA-containing trimers 26-28 inhibited HIV-1 replication to the sameextent as naturally occurring trimer 29, as determined by the inhibitionof HIV-1-induced syncytia formation. In contrast to the trimers 26-28,the Ade^(Bn)-containing trimers 22-25 inhibited HIV-1-induced syncytiaformation>1500-fold. On the other hand, the TCA-containing trimers 26-28inhibited HIV-1 RT activity by 99.7, 99.7, and 99.5%, respectively;however, the inhibition of HIV-1 RT activity by the Ade^(Bn)-containingtrimers 22-25 was dependent on the position of the Ade^(Bn) group in theoligonucleotide chain. The trimer 22, being N⁶-benzyl-substituted at the5′-terminus, inhibited HIV-1 RT activity by 33% compared to the trimers23-25, which inhibited HIV-1 RT activity by 7.6, 16.7, and 10.6%. TheTCA- and Ade^(Bn)-containing (2′-5′)trimers inhibited recombinant humanGST-RNase L activity as a function of the change in structure of thebase moiety. The (2′-5′)trimer 26 with the TCA moiety at the 5′-terminusactivated GST-RNase L by 87.7%, compared to 50% hydrolysis ofpoly(U)-3′-[³²P]pCp with naturally occurring trimer 29. The(2′-5′)trimers 22-25 with the Ade^(Bn) moiety instead of adenineactivated GST-RNase L by 37.4, 34.8, O, and 13.6%, respectively. Thesedata indicate that the adenine moiety at the 2′,3′-terminus of the(2′-5′)oligoadenylate trimer core 29 is essential for the activation ofrecombinant human RNase L.

Chemical Synthesis of Core Compounds

The synthesis of the unphosphorlyated compounds of the present inventionis illustrated by the following non-limiting examples.

General

TLC: Precoated silica gel thin-layer sheets 60 F 254 from Merck. Prep.column chromatography (CC): silica gel (Merck 60, 63-200 μm).Ion-exchange chromatography: DEAE-Servacel 23-SS (Serva). M.p.:Gallenkamp melting-point apparatus; no correction. UV/VIS: SpecordUV-VIS (Carl Zeiss, Germany); Λ_(max) in mn (log ε). ¹H-NMR: BrukerWM-360; δ in ppm rel. to SiMe₄.

PREPARATION 1

N⁶-Benzyl-5′-O-(monomethoxytrityl)adenosine (2)

To a solution (“soln.”) of N⁶-benzyladenosine (1, 1.67 g, 4.67 mmol) inpyridine (17 ml), 4-methoxytrityl chloride (2 g, 6.54 mmol) was added atr.t. The mixture was stirred at room temperature (“r.t.”) for 20 h andthen added dropwise to a mixture of H₂O and ice (800 g). The precipitatewas filtered off, dissolved in CHCl₃ (150 ml), and washed with H₂O (2×40ml). The organic layer was dried (Na₂SO₄) and evaporated. The residuewas purified by CC (silica gel, 15×3.5 cm, CHCl₃, then CHCl₃/MeOH 20:1)and finally crystallized from EtOH: 2.5 g (85%) of 2. M.p 165-167°. UV(MeOH): 234 (4.21), 271 (4.30). ¹H-NMR ((D₆) DMSO): 8.40 (s, NH); 8.27,8.13 (2s, H—C(2) H—C(8)); 7.37-6.82 (m, 19 arom. H); 5.93 (dd,H—C(1′));5.55 (d, OH—C(2′)); 5.22 (d, OH—C(3′)); 4.70(s,PhCH₂); 4.36 (dd,H—C(2′)); 4.30 (m,H—C(3′)); 4.05 (m,H—C(4′)); 3.71 (s, MeO); 3.21 (d,2H—C(5′)). Anal. calc. for C₃₇H₃₅N₃O₃ (629.7: C 70.57, H 5.60, N 11.12;found: C 70.43, H 5.56, N 11.21.

PREPARATION 2

2′,3′-Di-O-benzoyl-N⁶-benzyl-5′-O-(monomethoxytrityl)adenosine (3),2′-O-Benzoyl-N⁶-benzyl-5′-O-(monomethoxytrityl)adenosine (4), and3′-O-Benzoyl-N⁶-benzyl-5′-O-(monomethoxytrityl)adenosine (5)

A soln. of benzoyl cyanide (0.27 g, 2.06 mmol) in MeCN (20 ml) was addedat r.t. within 30 min to a soln. of 2, Et₃N (2.9 ml), and4-(dimethylamnino)pyridine (DMAP; 50 mg) in MeCN (30 ml). The mixturewas stirred at r.t. for 18 h and evaporated. Purification by CC (silicagel, 30×3.5 cm, hexane/AcOET 4:1→1:4) gave, after drying under highvacuum, 0.5 g (38%) of 3, 0.15 g (13%) of 4, and 0.54 g (46%) of 5 ascolorless foam.

(3): UV(MeOH): 232 (4.60), 272 (4.36). ¹H-NMR ((D₆) DMSO): 8.53 (s, NH):8.38, 8.17 (2s, H—C(2), H—C(8)); 7.94-6.79(m,29 arom. H); 6.52(d,H—C(1′)); 6.47 (dd,H—C(2′)); 6.20 (dd,H—C(3′)); 4.72 (s,PhCH₂); 4.60(m, H—C(4′)); 3.68 (s, MeO); 3.43 (m, 2H—C(5′)). Anal. calc. forC₅₁H₄₃N₅₇O-(837.9): C 73.10, H 5.17, N 8.35; found: C 73.32, H 5.20, N.8.24.

(4): UV(MeOH): 233 (4.45), 272 (4.32): ¹H-NMR ((D₆) DMSO): 8.50 (s, NH);8.36, 8.20 (2s, H—C(2), H—C(8)); 8.07-6.83 (m, 24 arom. H); 6.37 (d,H—C(1′)); 6.09 (dd. H—C(2′)); 5.75 (d, OH—C(3′)); 4.90 (dd, H—C(3′));4.72 (s, PhCH₂); 4.22 (m, H—C(4′)); 3.71 (s, MeO); 3.30 (m, 2H—C(5′)).Anal. calc. for C₄₄H₃₉N₅O₆ (733.8): C 72.01, H 5.35, N 9.54; found C72.18, H 5.30, N 9.37.

(5): UV(MeOH): 233 (4.48), 272 (4.34). ¹H-NMR ((D₆) DMSO): 8.47 (s, NH):8.35, 8.14 (2s, H—C(2), H—C(8′)); 8.07-6.80 (m. 24 arom. H); 6.04 (d,H—C(1′)); 5.98 (d, OH—C(2′)); 5.64 (dd, H—C(3′)); 5.23 (dd, H—C(2′));4.72 (s, PhCH₂); 4.39 (m, H—C(4′)); 3.67 (s, MeO); 3.36 (m, 2H—C(5′)).Anal. calc. for C₄₄H₃₉N₅O₆ (733.8): C 72.01, H 5.35, N 9.54; found: C72.20, H 5.40, N 9.40.

PREPARATION 3

2′,3′-Di-O-Benzoyl-N⁶-benzyladenosine (6)

A soln. of 3 (0.1 g. 0.12 mmol) was stirred with 2% TsOH in CH₂Cl₂/MeOH7:3 (10 ml) for 10 min. The mixture was diluted with CHCl₃ (100 ml) andwashed with H₂O (2×50 ml). The org. phase was dried (Na₂SO₄) andevaporated and the crude product purified by CC (silica gel, 10×2.5 cm,CHCl₃). Amorphous solid. UV(MeOH): 232 (4.32), 272 (4.36), ¹H-NMR ((D₆)DMSO): 8.65 (s, NH); 8.49, 8.25 (2s, H—C(2), H—C(8)); 8.07-7.11 (m, 15arom. H); 6.54 (d, H—C(1′)); 6.30 (dd, H—C(2′)); 5.92 (dd, H—C(3′));5.86 (t. OH—C(5′)); 4.72 (s, PhCH₂); 4.55 (m, H—C(4′)); 3.83 (m,2H—C(5′)); Anal. calc. for C₃₁H₂₇N₅O₆ (565.5: C 65.83, H 4.81, N 12.38;found: C 65.91 H 4.85, N 12.29.

PREPARATION 4

1-[5-O-(Monomethoxytrityl)-β-D-ribofuranosyl]-1H-1,2,4-triazole-3-carboxamide(8).

A mixture of ribavirin (7; 1 g, 4.1 mmol) and 4-methoxytrityl chloride(1.5 g, 4.9 mmol) in pyridine (50 ml) was stirred at r.t. for 48 h,evaporated, and co-evaporated with toluene (2×30 ml). The residue wasdissolved in CHCl₃ (100 ml) and washed with H₂O (2×50 ml). The org.layer was dried (Na₂SO₄), evaporated to a small volume (ca. 7 ml), andprecipitated with hexane to give, after drying under high vacuum, 1.95 g(92%) of 8. UV (MeOH); 231 (4.27). ¹H-NMR ((D₆) DMSO): 8.83 (s, H—C(5));7.75, 7.63 (2s, NH₂); 7.36-6.83 (m, 10 arom. H): 5.94 (d, H—C(1)), 5.65(d. OH—C(2′)); 5.20 (d, OH—C(3′)); 4.20 (m, H—C(2′)); 4.31 (m, H—C(3′));4.07 (m. H—C(4′)); 3.73 (s, MeO); 3.13 (m, 2H—C(5′)). Anal. calc. forC₂₈H₂₈N₄O₆ (516.5): C 65.10, H 5.46, N 10.84; found: C 65.30, H 5.30, N10.79.

PREPARATION 5

1-[2,3-Di-O-Benzoyl-5-O-(monomethoxytrityl)-β-D-ribofuranosyl]-1H-1,2,4-triazole-3-carboxamide(9) and1-[3-O-Benzoyl-5-O-monomethoxytrityl)-β-D-ribofuranosyl]-1H-1,2,4-triazoleCarboxamide (10)

To a soln. of 8 (1.85 g, 3.48 mmol) in MeCN (40 ml) Et₃N(6.3 ml), andDMAP (32 mg, 0.26 mmol): a soln. of benzoyl cyanide (0.55 g, 4.18 mmol)in MeCN (10 ml) was added dropwise within 3 h. The mixture was stirredat r.t. for 18 h and evaporated. Purification by CC (silica gel, 20×3.5cm, hexane/AcOEt 3:1→AcOEt) gave, after drying under high vacuum, 0.4 g(16%) of 9 and 1.3 g (57% of 10 as colorless foams.

(9): UV (MeOH): 231 (4.63). ¹H-NMR ((D₆)DMSO):8.94 (s, H—C(5));7.93-6.78 (m, 26H, NH₂, arom. H); 6.65 (d, H—C(1′)); 6.10 (dd, H—C(2′));6.03 (dd. H—C(3′)); 4.60 (m, H—C(4′)); 3.69 (s, MeO); 3.43 (m,2H—C(5′)). Anal. calc. for C₄₂H₃₆N₄O₈ (724.85): C 69.60, H 5.00, N 7.73;found: C 69.35, H 4.89, N 17.85.

(10): UV (MeOH): 231 (4.47). ¹H-NMR ((D₆) DMSO): 8.93 (s, H—C(5));8.04-6.81 (m, 21H, NH₂, arom. H); 6:12 (d, OH—C(2′)); 6.09 (d, H—C(1′));5.54 (dd, H—C(3′)); 4.90 (dd, H—C(2′)); 4.43 (m, H—C(4′)); 3.70 (s,MeO); 3.30 (m, 2H—C(5′)). Anal. calc. for C₃₅H₃₂N₄O₇ (620.7): C 67.73, H5.19, N 9.02; found: C 67.48, H 5.10, N 8.94.

PREPARATION 6

1-(2,3-Di-O-benzoyl-β-D-ribofuranosyl)-1H-1,2,4,-triazole-3-carboxamide(11)

A soln. of 9 (0.36 g, 0.5 mmol) in 80% AcOH (30 ml) was stirred at 50°for 15 min and evaporated. The residue was co-evaporated with EtOH (2×30ml) and crystallized from EtOH: 176 mg (78%) of 11. M.p. 172-173°. UV(MeOH):229 (4.54). ¹H-NMR (D₆) DMSO): 8.95 (s, H—C(5)); 7.95-7.40 (m,12H, NH₂, arom. H); 6.57 (d, H—C(1′)); 6.05 (dd, H—C(2′); 5.87 (dd,H—C(3′)); 4.55 (dd, H—C(4′)); 3.75 (m, 2H—C(5′)). Anal. calc. forC₂₂H₂₀N₄O₇ (452.4): C 58.40, H 4.45, N 12.38; found: C 58.20, H 4.32, N.12.27.

PREPARATION 7

3′-O-Benzoyl-N⁶-benzyl-5′-O-(monomethoxytrityl)adenosine2-[2-(4-Nitrophenyl)ethyl Trimethylammonium Phosphate] (13)

To a soln. of 1H-1,2,4-triazole (92 mg, 1.33 mmol) in pyridine (1.3 ml),2-chlorophenyl phosphorodichloridate (160 mg, 0.65 mmol) was added.After at r.t. for 10 min, the mixture was cooled with ice, and a soln.of 5 (0.32 g, 0.44 mmol) in pyridine (0.9 ml) was added. After 3 h,2-(4-nitrophenyl)ethanol (0.54 g, 3.25 mmol) was added and the mixturestirred at r.t for 18 h., diluted with CHCl₃ (100 ml), and washed with0.05M (Et₃NH)HCO₃ (2×50 ml). The org. phase was dried (Na₂SO₄),evaporated, and co-evaporated with toluene (2×20 ml). The residue wasdissolved in a soln. of 4-nitrobenzaldoxim (0.72 g, 4.33 mmol) indioxane/Et₃N/H₂O 1:1:1 (30 ml). After stirring at 4° for 20 h, themixture was evaporated and the residue purified by CC (silica gel,10×2.5 cm, CHCl₃/MeOH/Et₃N 95:4:1) 0.38 g (82%) of 13. Colorless foam.UV (MeOH): 233 (4.47), 272 (4.46). H¹-NMR (D₆) DMSO): 8.49 (s, NH);8.32, 8.13 (2s, H—C(2), H—C(8)); 8.03-6.79 (m, 28 arom. H); 6.27 (d,H—C(1′)); 5.86 (dd, H—C(3′); 5.65 (m, H—C(2′)); 4.70 (s, PhCH₂); 4.39(m, H—C(4′)); 3.68 (s, MeO). Anal. calc. for C₅₈H₆₂N₇O₁₁P (1064.1): C65.46, H 5.87, N 9.12 found: C 65.58, H 5.78, N. 8.97.

PREPARATION 8

1-[3-O-Benzoyl-5-O-(monomethoxytrityl)-β-D-ribofuranosyl]-1H-1,2,4-triazole-3-carboxamide2′-[2-(4-Nitrophenyl)ethyl Triethylammonium Phosphate] (14)

To a mixture of 1H-1,2,4-triazole (0.16 g, 2.38 mmol) and2-chlorophenylphosphorodichloridate (0.27 g, 1.19 mmol) in pyridine (2.2ml), a soln. of 10 (0.5 g, 0.81 mmol) in pyridine was added dropwise for15 min at +4°. After 3 h stirring at r.t., 2-(4-nitrophenyl)ethanol(0.54 g, 3.23 mmol) was added. The mixture was stirred at r.t. for 18 h,diluted with ChCl₃ (200 ml), and washed with 0.05M (Et₃NH)HCO₃ ( 2×100ml). The org. phase was dried (Na₂SO₄), evaporated, and co-evaporatedwith toluene (2×50 ml). The residue was dissolved in a soln. of4-nitrobenzaldoxime (0.45 g, 2.71 mmol) in dioxane/Et₃N/H₂O 1:1:1 (18ml). The mixture was stirred at r.t. for 24 h, evaporated, andco-evaporated with toluene (2×20 ml). The residue was purified by CC(silica gel, 10×2.5 cm, CHCl₃, and then CHCl₃/MeOH/Et₃N 95:4:1): 0.37 g(48%) of 14. Colorless foam. UV (MeOH): 230 (4.39), 273(4.07). H¹-NMR(D₆) DMSO): 8.94 (s, H—C(5)); 8.05-6.75 (m, 25H, NH₂, arom. H); 6.30(d,H—C(1′)); 5.78,(dd, H—C(3′)); 5.61 (m, H—C(2′)); 4.45 (m, H—C(4′));4.25 (m, OCH₂CH₂Ph); 3.69 (s, MeO); 3.31 (m, 2H—C(5′)); 2.81 (m,OCH₂CH₂Ph). Anal. calc. for C₄₉H₅₅N₆O₁₂P (951.0): C 61.88, H 5.82, N8.83 found: C 61.71, H 5.70, N. 8.69

PREPARATION 9

N⁶,3′-O-Dibenzoyladenylyl-{2′-{O^(P)-[2-(4-nitrophenyl)ethyl]}→5′}-2′,3′-di-O-benzoyl-N⁶-benzyladenosine (16)

A mixture of 6 (56 mg, 0.1 mmol), 15 (151 mg, 0.14 mmol), 1H-tetrazole(59 mg, 0.84 mmol), and TpsCl (85 mg, 0.28 mmol) in pyridine (1 ml) wasstirred at r.t for 16 h, diluted with CHCl₃, (50 m), and washed with0.05M (Et₃NH)HCO₃ (2×15 ml). The org. phase was dried (Na₂SG₄),evaporated, and co-evaporated with toluene (2×15 ml). The residue wasdissolved in 2% TsOH soln. (10 ml), and after 10 min, diluted with CHCl₃(50 m), and washed with 0.05M (Et₃NH)HCO₃. The org layer was dried(Na₂SO₄) and evaporated. The residue was purified by CC (silica gel,10×2.5 cm, CHCl₃): 105 mg (85%) of 16. Colorless foam. UV (MeOH): 234(4.70), 272 (4.66). Anal. calc. for C₆₃H₅₄N₁₁O₁₆P (1252.2): C 60.43, H4.34, N 12.30 found: C 60.59, H 4.42, N. 12.18.

PREPARATION 10

3′-O-Benzoyl-N⁶benzyladenylyl-{2′-{O^(P)[2-(4-nitrophenyl)ethyl]}→5′}-2′,3′-di-O-benzoyl-N⁶-benzyladenosine(17)

As described for 16, with 6 (40 mg, 0.071 mmol), 13 (105 mg, 0.1 mmol)pyridine (0.7 ml), TpsCl (60 mg, 0.188 mmol), 1H-tetrazole (42 mg, 0.59mmol), 2% TsOH soln. (5.7 ml), and 0.05M (Et₃NH)HCO₃. CC (silica gel,10×2.5 cm, CHCl₃) gave 71 mg (81%) of 17. Colorless foam. UV (MeOH): 234(4.71), 272 (4.65). Anal. calc. for C₆₃H₅₆N₁₁O₁₅P (1238.2): C 61.11, H4.55, N 12.44 found: C 61.30, H 4.60, N 12.23.

PREPARATION 11

3′-O-Benzoyl-N⁶-adenylyl-{2′-{O^(P)-[2-(4-nitrophenyl)ethyl]}→5′}-N⁶,2′-O,3′-O-tribenzoyladenosine (18)

As described for 16, with 12 (40 mg, 0.069 mmol), 13 (102 mg, 0.096mmol), pyridine (0.7 ml), TpsCl (60 mg, 0.188 mmol), 1H-tetrazole (42mg, 0.59 mmol), 2% TsOH soln. (5.5 ml), and 0.05M (Et₃NH)HCO₃. CC(silica gel, 10×2.5 cm, CHCl₃) gave 75 mg (87%) of 18. Colorless foam.UV (MeOH):234 (4.70), 272 (4.66). Anal. calc. for C₆₃H₅₄N₁₁O₁₆P(1252.2): C 60.43, H 4.34, N 12.30; found: C 60.55, H 4.29, N 12.19.

PREPARATION 12

N⁶,3′-O-Dibenzoyladenylyl-{2′-{O^(P)-[2-(4-nitrophenyl)ethyl]}→5′}-1-(2,3-di-O-benzoyl-β-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide(19)

As described for 16, with 11 (70 mg, 0.15 mmol), 15 (0.2 g, 0.18 mmol),pyridine (2 ml), TpsCl (0.17 g, 0.56 mmol), 1H-tetrazole (80 mg, 1.14mmol), 2% TsOH soln. (10 ml), and 0.05M (Et₃NH)HCO₃. CC (silica gel,10×2.5 cm, CHCl₃→CHCl₃/MeOH 19:1) gave 0.13 g (74%) of 19. Colorlessfoam. UV (MeOH):233 (4.72), 272 (4.35). Anal. calc. for C₅₄H₄₇N₁₀O₁₇P(1139.0): C 56.94, H 4.15, N 12.29; found: C 57.07, H 4.21, N 12.14.

PREPARATION 13

[1-(3-O-Benzoyl-β-D-ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide]yl-{2′-{O^(P)-[2-(4-nitrophenyl)ethyl]}→5′}-N⁶,2′-O,3′-O-tribenzoyladenosine(20)

As described for 16, with 12 (58 mg. 0.1 mmol), 14 (144 mg, 0.15 mmol),pyridine (2 ml) TpsCl (91 mg, 0.3 mmol), 1H-tetrazole (63 mg, 0.9 mmol).2% TsOH soln. (10 ml), and 0.05M (Et₃NH)HCO₃. CC silica gel, 9×2.5 cm,CHCl₃→CHCl₃/MeOH 19:1) gave 86 mg (76%) of 20. Colorless foam. UV(MeOH):233 (4.71), 273 (4.36). Anal. calc. for C₅₄H₄₇N₁₀O₁₇P (1139.0):C56.94, H 4.15, N 12.19; found: C 57.11, H 4.14, N 12.20.

EXAMPLE 1

Adenylyl-(2′-5′)-adenylyl-(2′-5′)-N⁶-benzyladenosineBis(triethylammonium) Salt (22)

A mixture of 15 (127 mg, 0.12 mmol) and 16 (105 mg, 0.08 mmol) inpyridine (0.8 ml), in the presence of Tps Cl (71 mg, 0.23 mmol) and1H-tetrazole (49 mg, 0.7 mmol), was stirred at r.t. for 18 h, dilutedwith CHCl₃ (50 ml), and washed with 0.05M (Et₃NH)HCO₃ 2×20 ml). The org.layer was dried (Na₂SO₄), evaporated, and co-evaporated with toluene(2×10 ml). The residue was treated with 2% TsOH soln. (8 ml), stirredfor 10 min, diluted with ChCl₃ (50 ml), and washed with 0.05M(Et₃NH)HCO₃ (2×15 ml). The org. layer was dried (Na₂SO₄) and evaporated.The residue was dissolved in 0.5M DBU in pyridine (16.4 ml) and stirredat r.t. for 18 h. Then the soln. was neutralized with 1M AcOH inpyridine (8.2 ml)and evaporated. The residue was dissolved in sat.NH₃/MeOH (40 ml), stirred at r.t. for 18 h, and evaporated, and theresidue taken up in CHCl₃/H₂O 1:1(100 ml). The org. phase was applied toan ion-exchange DEAE-Servacel-23-SS column (20×1.5 cm, linear gradientof 0.005→0.2M (Et₃NH)HCO₃ buffer (pH 7.5). The product fractions wereevaporated, and co-evaporated with MeOH (3×30 ml). The residual Et₃NH⁺salt was lyophilized (H₂O): 54 mg (53%) of 22. UV(H₂O). 263 (4.52).¹H-NMR (D₂O, t-BuOH as internal standard):6.91, 6.83, 6.73, 6.71,6.62,6.51 (6s, H—C(2), H—C(8)); 6.13 (m, 5 arom. H); 4.80, 4.67, 4.60 (3d,3H—C(1′)).

EXAMPLE 2

Adenylyl-(2′-5′)-N⁶-benzyladenylyl-(2′-5′)-adenosineBis(triethylammonium) Salt (23)

As described for 22, with 15 (91 mg. 0.08 mmol), 18 (75 mg, 0.06 mmol),pyridine (0.6 ml), TpsCl (51 mg, 0.17 mmol), 1H-tertrazole (35 mg, 0.45mmol), 2% TsOH soln. (5 ml), 0.5M DBU in pyridine (13.2 ml) 1M AcOH inpyridine (6.6 ml), and sat. NH₃ in MeOH (15 ml). Treatment withCHCl₃/H₂O 1:1 (100 ml) gave, after ion exchange (DEAE-Servacel 23-SS),49 mg (67%) of 23. UV (H₂O):263 (4.52), ¹H-NMR (D₂O, t-BuOH as internalstandard): 6.86, 6.81 (2H), 6.77, 6.56, 6.39 (5s, H—C(2), H—C(8)); 6.08(m, 5 arom. H); 4.77, 4.73, 4.57 (3d, 3H—C(1′)).

EXAMPLE 3

N⁶-Benzyladenylyl-(2′-5′)-adenylyl-(2′-5′)-adenosineBis(triethylammonium) Salt (24)

As described for 22, with 13 (32 mg, 0.03 mmol), 21 (32 mg, 0.025 mol),pyridine (0.3 ml), TpsCl (18 mg, 0.06 mmol), 1H-tetrazole (17 mg, 0.24mmol), 2% TsOH soln. (3 ml), ),5M DBU in pyridine (3.2 ml), 1M AcOH inpyridine (1.6 ml), and sat. NH₃ in MeOU (8 ml). Treatment with CHCl₃/H₂O1:1 (80 ml) gave, after ion exchange (DEAE-Servacel 23-SS), 16 mg (52%)of 24. UV (H₂O): 263 (4.48). ¹H-NMR (D₂O, t-BuOH as internal standard):6.92, 6.85, 6.73, 6.77 (2H), 6.50, (5s, H—C(2), H—C(8)); 6.07 (m, 5arom. H); 4.85, 4.67, 4.59 (3d, 3H—C(1′)).

EXAMPLE 4

N⁶-Benzyladenylyl-(2′-5′)-N⁶-benzyladenylyl-(2′-5′)-N⁶-benzyladenosineBis(triethylammonium) Salt (25)

As described for 22, with 13 (85 mg, 0.08 mmol), 17 (71 mg, 0.06 mmol),pyridine (0.6 ml), TpsCl (48 mg, 0.16 mmol), 1H-tetrazole (34 mg, 0.48mmol), 2% TSOH soln. (4.5 ml, 0.5M DBU in pyridine (9 ml), 1M AcOH inpyridine (4.5 ml), and sat. NH₃ in MeOH (30 ml). Treatment withCHCl₃/H₂O 1:1 (100 ml) gave, after ion exchange (DEAE-Servacel 23-SS),27 mg (76%) of 25. UV (H₂O): 270 (4.72). ¹H-NMR (D₂O, t-BuOH as internalstandard): 6.88, 6.83, 6.79, 6.73, 6.57, 6.50, (6s, H—C(2), H—C(8));6.04 (m, 15 arom. H); 4.82, 4.76, 4.62 (3d, 3H—C(1′)).

EXAMPLE 5

Adenylyl-(2′-5′)-adenylyl-(2′-5′)-1-(β-D-ribofuranosyl)-1H-1,2,4-triazole-3-carboxamideBis(triethylammonium) Salt (26)

As described for 22, with 15 (110 mg. 0.1 mmol), 19 (95 mg, 0.08 mmol),pyridine (0.9 ml), TpsCl (90 mg. 0.3 mmol), 1H-tertrazole (42 mg, 0.59mmol), 2% TsOH soln. (5 ml), 0.5M DBU in pyridine (10 ml) 1M AcOH inpyridine (5 ml), and sat. NH₃ in MeOH (30 ml). Treatment with CHCl₃/H₂O1:1 (100 ml) gave, after ion exchange (DEAE-Servacel 23-SS), 31 mg (34%)of 26. UV (H₂O):260 (4.42) ¹H-NMR (D₂O, t-BuOH as internal standard):7.13, 6.88, 6.83, 6.73, 6.45, (5s, H—C(2), H—C(5), H—C(8)); 4.85, 4.68,4.50 (3d, 3H—C(1′)).

EXAMPLE 6

Adenylyl-(2′-5′)-[1-(β-D-ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide]yl-(2′-5′)-adenosineBis(triethylammonium) Salt (27)

As described for 22, with 15 (86 mg, 0.08 mmol), 20 (57 mg, 0.05 mmol),pyridine (0.7 mnl), TpsCl (73 mg. 0.24 mmol), 1H-tertrazole (50 mg, 0.72mmol), 2% TsOH soln. (4 ml), 0.5M DBU in pyridine (7 ml) 1M AcOH inpyridine (3.5 mol), and sat. NH₃ in MeOH (25 ml). Treatment withCHCl₃/H₂O 1:1 (80 ml) gave, after ion exchange (DEAE-Servacel 23-SS), 13mg (24%) of 27. UV (H₂O):260 (4.42), ¹H-NMR (D₂O, t-BuOH as internalstandard): 7.06, 6.98, 6.90, 6.80, 6.71, (5s, H—C(2), H—C(5), H—C(8));4.87, 4.77, 4.62 (3d, 3H—C(1′)).

EXAMPLE 7

1-(β-D-Ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide]yl-(2′-5′)-adenylyl-(2′-5′)-adenosineBis(triethylammonium) Salt (28)

As described for 22, with 14 (76 mg. 0.08 mmol), 21 (63 mg, 0.05 mmol),pyridine (0.8 ml), TpsCl (73 mg, 0.24 mmol), 1H-tertrazole (50 mg, 0.72mmol), 2% TsOH soln. (5 ml), 0.5M DBU in pyridine (8 ml) 1M AcOH inpyridine (4 ml), and sat. NH₃ in MeOH (30 ml). Treatment with CHCl₃/H₂O1:1 (100 ml) gave, after ion exchange (DEAE-Servacel 23-SS), 26 mg (48%)of 28. UV (H₂O):260 (4.43), ¹H-NMR (D₂O, t-BuOH as internal standard):7.06, 6.88 (2H), 6.75, 6.65 (4s, H—C(2), HC(5), H—C(8)); 4.84, 4.73,4.59 (3d, 3H—C(1′)).

Phosphorylation of Core Compounds

The core compounds of the present invention may be 5′-monophosphorylatedaccording to the procedure of Sambrook el al., Molecular Cloning—ALaboratory Manual, 2 ed., Cold Spring Harbor Laboratory Press, pp.5.68-5.71 (1989) with ATP with T4 polynucleotide kinase.5′-Monophosphorylation may be determined by reverse-phase HPLC analysisand confirmed by the subsequent hydrolysis of each 5′-monophosphatederivative by 5′-nucleotidase. Yields of phosphorylation range from 15%to 60%. In the case where the R₂ groups of all internucleotide bonds(Formula I) of the molecule comprise oxygen, i.e., the linkages comprisephosphodiester bonds, the 5′-monophosphates are readily prepared byreacting the corresponding unphosphorylated core compound with POCl₃.

The 5′-diphosphate and 5′-triphosphate of the core compounds of theinvention may be prepared by following the procedure of Example 8.

EXAMPLE 8

All reactions are performed in glassware oven-dried at 125° C. for 18-24hr. Core compound (400 OD units at 260 nm) is dissolved in 500microliters of dry dimethylformamide (“DMF”) and dried in vacuo in a 10ml conical flask at 35° C. This process is repeated three times. To thedry residue, 50 micromoles of triphenylphosphine, 100 micromoles ofimidazole and 50 micromoles of dipyridinyl disulfide are added. Themixture is dissolved in 500 microliters dry DMF plus 50 microliters ofdry dimethylsulfoxide. The solution is stirred with a stirring bar for 2hr at room temperature. After 2 hr the solution is homogeneous (after 30minutes, the solution begins to change to yellow). The solution istransferred dropwise to 10 ml of a 1% NaI/dry acetone (w/v) solution.The clear colorless precipitate which forms is the sodium salt of the5′-phosphoroimidazolidate. The precipitate is centrifuged at roomtemperature, the supernatant is decanted, and the precipitate is washedthree times with 10 ml dry acetone. The centrifuging is repeated. Theprecipitate is dried over P₂O₅ in vacuo for 2 hr. The precipitate isdissolved in 200 microliters of freshly prepared 0.5 M tributylammoniumpyrophosphate in dry DMF. The solution is maintained at room temperaturefor 18 hr after which time the DMF is removed in vacuo. The residue isdissolved in 0.25M triethylammonium bicarbonate buffer (“TEAB”) (pH7.5). The 5′-di and 5′-triphosphate products are separated using aDEAE-Sephadex A25 column (HCO₃-form; 1×20 cm) with a linear gradient of0.25 M to 0.75 M TEAB. Fractions (10 ml) are collected. The product isobserved by ultraviolet spectroscopy at 254 mn. The fractions containingthe 5′-di and 5′-triphosphates are separately pooled and dried in vacuo.The TEAB is removed by repeated addition of water followed bylyophilization. The yield of the 5′-diphosphate is about 5%; the yieldof the 5′-triphosphate is about 60%.

All references cited with respect to synthetic, preparative andanalytical procedures are incorporated by reference.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof and,accordingly, reference should be made to the appended claims, ratherthan to the foregoing specification, as indication the scope of theinvention.

REFERENCES

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What is claimed is:
 1. A compound of the formula:

wherein m is zero, 1, 2 or 3; n is from 1 to 8; R is independentlyselected from the group consisting of

 provided that all R may not be

R₁ is independently selected from the group consisting of hydroxyl andhydrogen; R₂ is independently selected from the group consisting ofoxygen and sulfur; or water-soluble salt of said compound.
 2. A compoundaccording to claim 1 wherein m is
 3. 3. A compound according to claim 1wherein m is
 1. 4. A compound according to claim 1 wherein m is
 0. 5. Acompound according to any of claims 1, 2, 3 or 4 wherein n is 1 or
 2. 6.A compound according to claim 5 wherein each R₁ is hydroxyl and each R₂is oxygen.
 7. A compound according to clain 6 wherein each R is selectedfrom the group consisting of


8. A compound according to claim 6 wherein each R is selected from thegroup consisting of


9. A compound according to claim 7 selected from the group consisting ofadenylyl-(2′-5′)adenylyl-(2′-5′)-1-(β-D-ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide,the 5′-mono-, di-, and triphosphates thereof, and water-soluble salts ofany of them.
 10. A compound according to claim 7 selected from the groupconsisting ofadenylyl-(2′-5′)-[1-(β-D-ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide]yl2′-5′)-adenosine,the 5′-mono-, di-, and triphosphates thereof, and water-soluble salts ofany of them.
 11. A compound according to claim 7 selected from the groupconsisting of1-(β-D-ribofaranosyl)-1H-1,2,4-triazole-3-carboxamide]-(2′-5′)-adenylyl-(2′-5′)-adenosine,the 5′-mono-, di-, and triphosphates thereof, and water-soluble salts ofany of them.
 12. A compound according to claim 8 selected from the groupconsisting of adenylyl-(2′-5′)-adenylyl-(2′-5′)-N⁶-benzyladenosine, the5′-mono-, di-, and triphosphates thereof, and water-soluble salts of anyof them.
 13. A compound according to claim 8 selected from the groupconsisting of adenylyl-(2′-5′)-N⁶-benzyladenylyl-(2′-5′)-adenosine, the5′-mono-, di-, and triphosphates thereof, and water-soluble salts of anyof them.
 14. A compound according to claim 8 selected from the groupconsisting of N⁶-benzyladenylyl-(2′-5′)-adenylyl-(2′-5′)-adenosine, the5′-mono, di-, and triphosphates thereof, and water-soluble salts of anyof them.
 15. A compound according to claim 8 selected from the groupconsisting ofN⁶-benzyladenylyl-(2′-5′)-N⁶benzyladenylyl-(2′-5′)-N⁶-benzyladenosine,the 5′-mono-, di-, and triphosphates thereof, and water-soluble salts ofany of them.
 16. A compound or water-soluble salt according to any ofclaims 9, 10, 11, 12, 13, 14 or 15 wherein m is zero.
 17. An antiviralcomposition comprising a compound or water-soluble salt thereofaccording to any of claim 1 in combination with an agricultural carrier.18. An antiviral composition comprising a compound or water-soluble saltthereof according to any of claim 1 in combination with a pharmaceuticalcarrier.
 19. A method of treating viral infection in a plant comprisingadministering thereto an antiviral effective amount of a compound orwater-soluble salt thereof according to any of claim
 1. 20. A method oftreating viral infection in a mammal comprising administering thereto anantiviral effective amount of a compound or water-soluble salt thereofaccording to any of claim 1.